Degradation of hydrophobic ester pesticides and toxins

ABSTRACT

The present invention relates to methods for degrading hydrophobic ester pesticides and toxins. In particular, the present invention relates to the use of insect esterases, and mutants thereof, in the bioremediation of hydrophobic ester pesticides and toxins residues, such as pyrethroid residues, contaminating the environment and horticultural commodities.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.10/503,643, filed on Mar. 23, 2005, as a 371 application of PCTAU2002/00114, filed Feb. 6, 2002.

FIELD OF THE INVENTION

This invention relates to enzymes and methods for degrading hydrophobicester pesticides and toxins. In particular, the present inventionrelates to the use of insect esterases, such as α-carboxylesterases, andmutants thereof, in the bioremediation of pyrethroid residuescontaminating the environment and horticultural commodities.

BACKGROUND OF THE INVENTION

Pyrethroids constitute a major class of chemical pesticides. They aresynthetic analogues of the natural pyrethrins, which are produced in theflowers of the pyrethrum plant (Tanacetum cinerariifolium). Modificationof their structure has yielded compounds that retain the intrinsicallymodest vertebrate toxicity of the natural products but are both morestable and more potent as pesticides. In the thirty years since theirintroduction they have risen to about 10-20% of insecticide salesworldwide and they are projected to retain substantial market share intothe foreseeable future. They are now widely used across agriculturalproduction and processing systems in many countries and have causedresidue incidents in diverse commodities ranging from cotton andhorticulture through to wool.

Residues of pyrethroid pesticides are undesirable contaminants of theenvironment and a range of commodities. They are undesirable because ofthe broad target range of the pesticide across invertebrates and theirsignificant toxicity to vertebrates, although they are generallyconsidered to be amongst the safest pesticides to mammals. Areas ofparticular sensitivity include contamination of soil, irrigationtailwater that is re-cycled, used by irrigators downstream or simplyallowed to run off-farm, and residues above permissible levels inhorticultural exports. Animal industries also have problems withpesticide-contaminated commodities arising through either their ownpesticide use or their reliance on crop products and by-products asfodder. Processing wastes from food processing plants, carpet dye bathsand animal dips are also contaminated, sometimes quite heavily, withpesticide residues. Bioremediation strategies are therefore required foreliminating or reducing these pesticide residues.

One proposed bioremediation strategy involves the use of enzymes capableof immobilising or degrading the pesticide residues. Such enzymes may beemployed, for example, in bioreactors through which contaminated watercould be passed, or in washing solutions after post-harvestdisinfestation of fruit, vegetables or animal products to reduce residuelevels and withholding times. Suitable enzymes for degrading pesticideresidues include OP hydrolases from bacteria, vertebrates andorganophosphate (OP) resistant insects. It is desirable that thehydrolytic enzymes degrade the pesticide residues at a rapid rate.

Organophosphate resistance in the sheep blowfly, Lucilia cuprina, isconferred by two different mutations in the gene encodingcarboxylesterase E3. The two mutant enzymes differ in their substratespecificities but between them can detoxify two major subtypes of OPs.The E3 gene from L. cuprina was cloned by Newcomb et al. (1997) and,using a combination of DNA sequencing, baculovirus expression and invitro mutagenesis, these workers identified the two resistancemutations. One is an Asp for Gly substitution at residue 137 in theoxyanion hole region of the active site (Newcomb et al., 1997). Theother is a Leu for Trp substitution at residue 251 in thesubstrate-binding region (Campbell et al., 1998), which results in anincrease in malathion carboxylesterase activity as well as theacquisition of OP hydrolase activity.

There is a need for methods and enzymes which can be used for thebioremediation of, for example, soils, foodstuff and water samplescontaminated with hydrophobic ester pesticides and toxins.

SUMMARY OF THE INVENTION

The present inventors have now found that insect esterases, and mutantsthereof, are able to hydrolyse hydrophobic ester pesticides and toxinssuch as pyrethroids. The activity of the insect esterases, and mutantsthereof, show a degree of chiral specificity, which differed betweenmutants. It is therefore possible to provide a suite of insectesterases, or mutants thereof, that are able to degrade hydrophobicester pesticides and toxins that can act, alone or together, aseffective bioremediation agents for hydrophobic ester pesticides andtoxins such as pyrethroids.

Accordingly, in a first aspect, the present invention provides a methodof eliminating or reducing the concentration of a hydrophobic esterpesticide or toxin in a sample, the method comprising contacting thesample with an insect esterase, or a mutant thereof.

In a preferred embodiment of the first aspect, the insect esterase is amember of the carboxyl/cholinesterase multi-gene family of enzymes. Morepreferably, the insect esterase is an α-carboxylesterase. Even morepreferably, the insect esterase is a member of the α-carboxylesterasecluster which forms a sub-clade within this multi-gene family (Oakeshottet al., 1999). Esterases which form this sub-clade include at leastα-carboxylesterases which can be isolated from species of Diptera,Hemiptera and Hymenoptera. Specific enzymes which are found in thissub-clade include, but are not limited to, the E3 or EST23 esterases.However, orthologous esterases of E3 and EST23 from other insect speciescan also be used in the methods of the present invention.

Preferably, the α-carboxylesterases can be isolated from a species ofDiptera. Accordingly, examples of preferred α-carboxylesterases for usein the present invention are the E3 esterase (SEQ ID NO:1) which isderived from Lucilia cuprina, or the EST23 esterase (SEQ ID NO:2) whichis derived from Drosophila melanogaster.

In a further preferred embodiment, the mutant insect esterase has amutation(s) in the oxyanion hole, acyl binding pocket or anionic siteregions of the active site, or any combination thereof.

In a further preferred embodiment, the mutant α-carboxylesterase isselected from the group consisting of: E3G137R, E3G137H, E3W251L,E3W251S, E3W251G, E3W251T, E3W251A, E3W251L/F309L, E3W251L/G137D,E3W251L/P250S, E3F309L, E3Y148F, E3E217M, E3F354W, E3F354L, andEST23W251L. Preferably, the mutant α-carboxylesterase is E3W251L,E3F309L, E3W251L/F309L or EST23W251L.

In another preferred embodiment of the first aspect, theα-carboxylesterase, or mutant thereof, has a sequence selected from thegroup consisting of:

i) a sequence as shown in SEQ ID NO:1,

ii) a sequence as shown in SEQ ID NO:2, and

iii) a sequence which is at least 40% identical to i) or ii) which iscapable of hydrolysing a hydrophobic ester pesticide or toxin. Morepreferably, the polypeptide is at least 50% identical, more preferablyat least 60% identical, more preferably at least 70% identical, morepreferably at least 80% identical, and more preferably at least 90%identical, more preferably at least 95% identical, and even morepreferably at least 97% identical to i) or ii).

As the skilled addressee would be aware, the method of the first aspectcan be performed using more than one insect esterase, or mutantsthereof. This is particularly the case where different insect esterases,or mutants thereof, have different hydrolytic activity for differentstereo-isomers of the hydrophobic ester pesticide or toxin.

The hydrophobic ester pesticide or toxin can be any molecule which ishydrophobic in nature, contains an ester group and has some level oftoxicity towards living organisms. A particularly preferred hydrophobicester pesticide or toxin is a pyrethroid. The pyrethroid can be a Type Ior Type II pyrethroid. Preferably, the Type I pyrethroid is selectedfrom the group consisting of: 1S/1R trans permethrin, 1S/1R cispermethrin, NRDC157 1S cis, and NRDC157 1R cis. Preferably, the Type IIpyrethroid is deltamethrin.

Preferably, the sample is a soil sample, a water sample or a biologicalsample. Preferred biological samples include matter derived from plantssuch as seeds, vegetables or fruits, as well as matter derived fromanimals such as meat.

Preferably, the method is performed in a liquid containing environment.

The sample can be exposed to the insect esterase, or mutant thereof, byany appropriate means. This includes providing the insect esterase, ormutant thereof, directly to the sample, with or without carriers orexcipients etc. The insect esterase, or mutant thereof, can also beprovided in the form of a host cell, typically a microorganism such as abacterium or a fungus, which expresses a polynucleotide encoding theinsect esterase, or mutant thereof.

The insect esterase, or mutant thereof, can also be as provided apolymeric sponge or foam, the foam or sponge comprising the insectesterase, or mutant thereof, immobilized on a polymeric porous support.

Preferably, the porous support comprises polyurethane.

In a preferred embodiment, the sponge or foam further comprises carbonembedded or integrated on or in the porous support.

It is envisaged that the use of a surfactant in the method of thepresent invention may liberate hydrophobic ester pesticides and/ortoxins from any, for example, sediment in the sample. Thus increasingefficiency of the method of the present invention. Accordingly, inanother preferred embodiment, the method comprises the presence of asurfactant when the hydrophobic ester pesticide or toxin is contactedwith the insect esterase, or mutant thereof. More preferably, thesurfactant is a biosurfactant.

Further, hydrophobic ester pesticide or toxin in a sample can also bedegraded by exposing the sample to a transgenic plant which produces theinsect esterase, or mutant thereof.

In a second aspect the present invention provides a substantiallypurified polypeptide which is a mutant of an insect esterase, whereinone or more mutations are within a region of the esterase selected fromthe group consisting of: oxyanion hole, acyl binding pocket and anionicsite, wherein the mutant insect esterase is capable of hydrolysing ahydrophobic ester pesticide or toxin, with the proviso that the mutantinsect esterase is not E3W251L, E3W251S, E3W251G or E3G137D.

Preferably, the insect esterase is an α-carboxylesterase.

Preferably, the polypeptide is selected from the group consisting of:

i) a mutant of a sequence as shown in SEQ ID NO:1, and

ii) a mutant of sequence as shown in SEQ ID NO:2, wherein the mutant isat least 40% identical to at least one of SEQ ID NO's:1 or 2. Morepreferably, the mutant is at least 80% identical to at least one of SEQID NO's:1 or 2. Even more preferably, the mutant is at least 90%identical to at least one of SEQ ID NO's:1 or 2.

Preferably, the mutation is a point mutation.

Preferably, the polypeptide selected from the group consisting of:E3G137R, E3G137H, E3W251T, E3W251A, E3W251L/F309L, E3W251L/G137D,E3W251L/P250S, E3F309L, E3Y148F, E3E217M, E3F354W, E3F354L, EST23W251L.

In a third aspect, the present invention provides a fusion polypeptidecomprising a polypeptide according to the second aspect fused to atleast one other polypeptide sequence.

In a fourth aspect the present invention provides an isolatedpolynucleotide encoding a polypeptide according to the second or thirdaspects.

In a fifth aspect the present invention provides a vector forreplication and/or expression of a polynucleotide according to thefourth aspect.

In a sixth aspect the present invention provides a host cell transformedor transfected with the vector of the fifth aspect.

In a seventh aspect the present invention provides a composition forhydrolysing a hydrophobic ester pesticide or toxin, the compositioncomprising a polypeptide according to the second or third aspects, andone or more acceptable carriers.

In an eighth aspect the present invention provides a method forgenerating and selecting an enzyme that hydrolyses a hydrophobic esterpesticide or toxin, the method comprising

(i) introducing one or more mutations into an insect esterase, or aninsect esterase that has already been mutated, and

(ii) determining the ability of the mutant insect esterase to hydrolysea hydrophobic ester pesticide or toxin.

Preferably, the one or more mutations enhances hydrolytic activityand/or alters the stereospecificity of the esterase.

Such one or more mutations can be introduced by a variety of techniquesknown to the skilled addressee. These techniques include, but are notlimited to, site directed mutagenesis, random mutagenesis, or the use ofDNA shuffling in in vitro evolution techniques, each of which areperformed on a polynucleotide encoding the insect esterase or insectesterase that has already been mutated.

In a preferred embodiment of the eighth aspect, the insect esterase isan α-carboxylesterase. More preferably, the α-carboxylesterase is an E3or EST23 esterase. More preferably, the α-carboxylesterase has asequence selected from the group consisting of:

i) a sequence as shown in SEQ ID NO:1,

ii) a sequence as shown in SEQ ID NO:2, and

iii) a sequence which is at least 40% identical to i) or ii). Morepreferably, the polypeptide is at least 50% identical, more preferablyat least 60% identical, more preferably at least 70% identical, morepreferably at least 80% identical, and more preferably at least 90%identical, more preferably at least 95% identical, and even morepreferably at least 97% identical to i) or ii).

Preferably, the one or more mutations are within a region of theesterase selected from the group consisting of: oxyanion hole, acylbinding pocket and anionic site.

In a further preferred embodiment, the insect esterase that has alreadybeen mutated is selected from the group consisting of: E3G137R, E3G137H,E3W251L, E3W251S, E3W251G, E3W251T, E3W251A, E3W251L/F309L,E3W251L/G137D, E3W251L/P250S, E3F309L, E3Y148F, E3E217M, E3F354W,E3F354L, and EST23W251L.

In a further preferred embodiment of the eighth aspect, the mutation isa point mutation.

In a ninth aspect the present invention provides an enzyme obtained by amethod according to the eighth aspect.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting example and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1: Amino acid sequence alignment of the E3 (SEQ ID NO:1) andTorpedo californica acetylcholinesterase (SEQ ID NO:3) enzymes. Thesequence around the active site serine and residues Gly137, Trp251 andPhe309 are shown in bold and underlined.

FIG. 2: Proposed configuration of active site of LcE3 carboxylesterasein an acylation reaction.

FIG. 3: Results of representative titration experiments performed oncell extracts containing baculovirus expressed esterases.

FIG. 4: Molecular structures for 1R/S cis and trans permethrin, 1R/S cisand trans NRDC157 and the four stereoisomers of cis deltamethrin.

FIG. 5: Hydrolysis of cis and trans permethrin (0.5 μM) by E3W251L.

KEY TO SEQUENCE LISTING

SEQ ID NO:1—Amino acid sequence of Lucilia cuprina E3α-carboxylesterase.

SEQ ID NO:2—Amino acid sequence of Drosophila melanogaster EST23α-carboxylesterase.

SEQ ID NO:3—Partial amino acid sequence of Torpedo californicaacetylcholinesterase.

DETAILED DESCRIPTION OF THE INVENTION General Techniques

Unless otherwise indicated, the recombinant DNA techniques utilized inthe present invention are standard procedures, well known to thoseskilled in the art. Such techniques are described and explainedthroughout the literature in sources such as, J. Perbal, A PracticalGuide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarbourLaboratory Press (1989), T. A. Brown (editor), Essential MolecularBiology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M.Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach,Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al.(Editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent) and are incorporated herein by reference.

Pyrethroids

Pyrethroids are synthetic analogs of pyrethrum pesticides. For example,pyrethroids include (in each case common name in accordance with ThePesticide Manual, 12th Edition): permethrin, fenvalerate, esfenvalerate,cypermethrin, alpha-cypermethrin, deltamethrin, fenpropathrin,fluvalinate, flucythrinate, cyfluthrin, acrinathrin, tralomethrin,cycloprothrin, lambda-cyhalothrin, tefluthrin, bifenthrin,transfluthrin, zeta-cypermethrin, and halfenprox.

Type I pyrethroid compounds (e.g., permethrin) differ from type IIpyrethroid compounds in that type II compounds possess a cyano group onthe α-carbon atom of the phenoxybenzyl moiety. Some examples of type IIpyrethroids are cypermethrin, deltamethrin, and fenvalerate.

Examples of pyrethroid pesticides which can be hydrolysed using themethods of the present invention include, but are not restricted tothese compounds; 3-phenoxybenzyl(1RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate[permethrin],α-cyano-3-phenoxybenzyl-1-(4-ethoxyphenyl)-2,2-dichlorocyclopropanecarboxylate [cyloprothrin],(RS)-α-cyano-3-phenoxybenzyl(RS)-2-(4-chlorophenyl)-3-isovalerate[fenvalerate],(S)-α-cyano-3-phenoxybenzyl(S)-2-(4-chlorophenyl)isovalerate[esfenvalerate],α-cyano-3-phenoxybenzyl(S)-2-(4-difluoromethoxyphenyl)isovalerate[flucythrinate], α-cyano-3-phenoxybenzyl2-(2-chloro-4-trifluoromethylaniline)isovalerate [fluvalinate],(RS)-α-cyano-3-phenoxybenzyl 2,2,3,3-tetramethylcyclopropane carboxylate[fenpropathrin], 3-phenoxybenzyl(1R)-cis,trans-chrysanthemate[d-fenothrin], (RS)-α-cyano-3-phenoxybenzyl(1R)-cis,trans-chrysanthemate[cyfenothrin],(RS)3-allyl-2-methyl-4-oxocyclopento-2-enyl(1RS)-cis,trans-chrysanthemate[allethrin],α-cyano-3-phenoxybenzyl(1R)-cis,trans-3-phenoxybenzyl(1R)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate [cypermethrin],(S)-α-cyano-3-phenoxybenzyl(1R)-cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate [deltamethrin],(S)-α-cyano-3-phenoxybenzyl(1R)-cis-2,2-dimethyl-3-(1,2,2,2-tetrabromoethyl)cyclopropanecarboxylate [tralomethrin], 3,4,5,6-tetrahydroimidomethyl(1RS)-cis,trans-chrysanthemate [tetramethrin],5-benzyl-3-furylmethyl(1RS)-cis,trans-chrysanthemate [resmethrin],α-cyano-4-fluoro-3-phenoxybenzyl(1R,trans)-2,2-dimethyl-3-(2,2-dichlorovinyl)cyclopropanecarboxylate [cyfluthrin].

Polypeptides

By “substantially purified” we mean a polypeptide that has beenseparated from most of the lipids, nucleic acids, other polypeptides,and other contaminating molecules with which it is associated in itsnative state.

The % identity of a polypeptide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 15 aminoacids in length, and the GAP analysis aligns the two sequences over aregion of at least 15 amino acids. More preferably, the query sequenceis at least 50 amino acids in length, and the GAP analysis aligns thetwo sequences over a region of at least 50 amino acids. More preferably,the query sequence is at least 100 amino acids in length and the GAPanalysis aligns the two sequences over a region of at least 100 aminoacids. More preferably, the query sequence is at least 250 amino acidsin length and the GAP analysis aligns the two sequences over a region ofat least 250 amino acids. Even more preferably, the query sequence is atleast 500 amino acids in length and the GAP analysis aligns the twosequences over a region of at least 500 amino acids.

As used herein, the term “mutant thereof” refers to mutants of anaturally occurring insect esterase which maintains at least somehydrolytic activity towards a hydrophobic ester pesticide or toxin whencompared to the naturally occurring insect esterase from which they arederived. Preferably, the mutant has enhanced activity and/or alteredstereospecificity when compared to the naturally occurring insectesterase from which they are derived.

Amino acid sequence mutants of naturally occurring insect esterases canbe prepared by introducing appropriate nucleotide changes into a nucleicacid of the present invention, or by in vitro synthesis of the desiredpolypeptide. Such mutants include, for example, deletions, insertions orsubstitutions of residues within the amino acid sequence. A combinationof deletion, insertion and substitution can be made to arrive at thefinal construct, provided that the final protein product possesses thedesired characteristics.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. In a particularly preferred embodiment, naturally occurringinsect esterases are mutated to increase their ability to hydrolyse ahydrophobic ester pesticide or toxin, particularly a pyrethroid. Thesites for mutation can be modified individually or in series, e.g., by(1) substituting first with conservative amino acid choices and thenwith more radical selections depending upon the results achieved, (2)deleting the target residue, or (3) inserting other residues adjacent tothe located site. Examples of such mutants include; E3G137R, E3G137H,E3W251L, E3W251S, E3W251G, E3W251T, E3W251A, E3W251L/F309L,E3W251L/G137D, E3W251L/P250S, E3F309L, E3Y148F, E3E217M, E3F354W,E3F354L, and EST23W251L.

Mutants useful for the methods of the present invention can also beobtained by the use of the DNA shuffling technique (Patten et al.,1997). DNA shuffling is a process for recursive recombination andmutation, performed by random fragmentation of a pool of related genes,followed by reassembly of the fragments by primerless PCR. Generally,DNA shuffling provides a means for generating libraries ofpolynucleotides which can be selected or screened for, in this case,polynucleotides encoding enzymes which can hydrolyse a hydrophobic esterpesticide or toxin. The stereospecificity of the selected enzymes canalso be screened.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. The sites of greatest interest for substitutional mutagenesisinclude sites identified as the active or binding site(s). Other sitesof interest are those in which particular residues obtained from variousstrains or species are identical. These positions may be important forbiological activity. These sites, especially those falling within asequence of at least three other identically conserved sites, can besubstituted in a relatively conservative manner. Such conservativesubstitutions are shown in Table 1 under the heading of “exemplarysubstitutions”.

Furthermore, if desired, unnatural amino acids or chemical amino acidanalogues can be introduced as a substitution or addition into theinsect esterase, or mutants thereof. Such amino acids include, but arenot limited to, the D-isomers of the common amino acids,2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid,2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cαmethyl amino acids, Nα-methyl amino acids, and amino acidanalogues in general.

Also included within the scope of the invention are insect esterases, ormutants thereof, which are differentially modified during or aftersynthesis, e.g., by biotinylation, benzylation, glycosylation,acetylation, phosphorylation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. These modifications may serve toincrease the stability and/or bioactivity of the polypeptide of theinvention.

TABLE 1 Original Exemplary Residue Substitutions Ala (A) val; leu; ile;gly Arg (R) lys Asn (N) gln; his Asp (D) glu Cys (C) ser Gln (Q) asn;his Glu (E) asp Gly (G) pro, ala His (H) asn; gln Ile (I) leu; val; alaLeu (L) ile; val; met; ala; phe Lys (K) arg Met (M) leu; phe Phe (F)leu; val; ala Pro (P) gly Ser (S) thr Thr (T) ser Trp (W) tyr Tyr (Y)trp; phe Val (V) ile; leu; met; phe, ala

Insect esterases, and mutants thereof, can be produced in a variety ofways, including production and recovery of natural proteins, productionand recovery of recombinant proteins, and chemical synthesis of theproteins. In one embodiment, an isolated polypeptide encoding the insectesterase, or mutant thereof, is produced by culturing a cell capable ofexpressing the polypeptide under conditions effective to produce thepolypeptide, and recovering the polypeptide. A preferred cell to cultureis a recombinant cell of the present invention. Effective cultureconditions include, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Aneffective medium refers to any medium in which a cell is cultured toproduce a polypeptide of the present invention. Such medium typicallycomprises an aqueous medium having assimilable carbon, nitrogen andphosphate sources, and appropriate salts, minerals, metals and othernutrients, such as vitamins. Cells producing the insect esterase, ormutant thereof, can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes, and petriplates. Culturing can be carried out at a temperature, pH and oxygencontent appropriate for a recombinant cell. Such culturing conditionsare within the expertise of one of ordinary skill in the art.

Polynucleotides

By “isolated polynucleotide”, we mean a polynucleotide separated fromthe polynucleotide sequences with which it is associated or linked inits native state. Furthermore, the term “polynucleotide” is usedinterchangeably herein with the term “nucleic acid molecule”.

The % identity of a polynucleotide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 45nucleotides in length, and the GAP analysis aligns the two sequencesover a region of at least 45 nucleotides. Preferably, the query sequenceis at least 150 nucleotides in length, and the GAP analysis aligns thetwo sequences over a region of at least 150 nucleotides. Morepreferably, the query sequence is at least 300 nucleotides in length andthe GAP analysis aligns the two sequences over a region of at least 300nucleotides.

Recombinant Vectors

Recombinant vectors can be used to express an insect esterase, or mutantthereof, for use in the methods of the present invention. In addition,in another embodiment of the present invention includes a recombinantvector, which includes at least one isolated polynucleotide molecule ofthe present invention, inserted into any vector capable of deliveringthe polynucleotide molecule into a host cell. Such vectors containheterologous polynucleotide sequences, that is polynucleotide sequencesthat are not naturally found adjacent to polynucleotide encoding theinsect esterase, or mutant thereof, and that preferably are derived froma species other than the species from which the esterase is derived. Thevector can be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a virus or a plasmid.

One type of recombinant vector comprises a polynucleotide encoding aninsect esterase, or mutant thereof, operatively linked to an expressionvector. The phrase operatively linked refers to insertion of apolynucleotide molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.As used herein, an expression vector is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of aspecified polynucleotide molecule. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors canbe either prokaryotic or eukaryotic, and are typically viruses orplasmids. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in recombinantcells of the present invention, including in bacterial, fungal,endoparasite, arthropod, other animal, and plant cells. Preferredexpression vectors of the present invention can direct gene expressionin bacterial, yeast, arthropod and mammalian cells and more preferablyin the cell types disclosed herein.

Expression vectors of the present invention contain regulatory sequencessuch as transcription control sequences, translation control sequences,origins of replication, and other regulatory sequences that arecompatible with the recombinant cell and that control the expression ofpolynucleotide molecules of the present invention. In particular,expression vectors which comprise a polynucleotide encoding an insectesterase, or mutant thereof, include transcription control sequences.Transcription control sequences are sequences which control theinitiation, elongation, and termination of transcription. Particularlyimportant transcription control sequences are those which controltranscription initiation, such as promoter, enhancer, operator andrepressor sequences. Suitable transcription control sequences includeany transcription control sequence that can function in at least one ofthe recombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin bacterial, yeast, arthropod and mammalian cells, such as, but notlimited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophagelambda, bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6,bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcoholoxidase, alphavirus subgenomic promoters (such as Sindbis virussubgenomic promoters), antibiotic resistance gene, baculovirus,Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoonpoxvirus, other poxvirus, adenovirus, cytomegalovirus (such asintermediate early promoters), simian virus 40, retrovirus, actin,retroviral long terminal repeat, Rous sarcoma virus, heat shock,phosphate and nitrate transcription control sequences as well as othersequences capable of controlling gene expression in prokaryotic oreukaryotic cells. Additional suitable transcription control sequencesinclude tissue-specific promoters and enhancers.

Polynucleotide encoding an insect esterase, or mutant thereof, may also(a) contain secretory signals (i.e., signal segment nucleic acidsequences) to enable an expressed insect esterase, or mutant thereof, tobe secreted from the cell that produces the polypeptide and/or (b)contain fusion sequences. Examples of suitable signal segments includeany signal segment capable of directing the secretion of an insectesterase, or mutant thereof. Preferred signal segments include, but arenot limited to, tissue plasminogen activator (t-PA), interferon,interleukin, growth hormone, histocompatibility and viral envelopeglycoprotein signal segments, as well as natural signal sequences. Inaddition, polynucleotides encoding an insect esterase, or mutantthereof, can be joined to a fusion segment that directs the encodedprotein to the proteosome, such as a ubiquitin fusion segment.

Host Cells

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more polynucleotidesencoding an insect esterase, or mutant thereof. Transformation of apolynucleotide molecule into a cell can be accomplished by any method bywhich a polynucleotide molecule can be inserted into the cell.Transformation techniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. A transformed polynucleotideencoding an insect esterase, or mutant thereof, can remainextrachromosomal or can integrate into one or more sites within achromosome of the transformed (i.e., recombinant) cell in such a mannerthat their ability to be expressed is retained.

Suitable host cells to transform include any cell that can betransformed with a polynucleotide encoding an insect esterase, or mutantthereof. Host cells of the present invention either can be endogenously(i.e., naturally) capable of producing an insect esterase or mutantthereof, or can be capable of producing such proteins after beingtransformed with at least one polynucleotide encoding an insectesterase, or mutant thereof. Host cells of the present invention can beany cell capable of producing at least one insect esterase, or mutantthereof, and include bacterial, fungal (including yeast), parasite,arthropod, animal and plant cells. Preferred host cells includebacterial, mycobacterial, yeast, arthropod and mammalian cells. Morepreferred host cells include Salmonella, Escherichia, Bacillus,Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK(baby hamster kidney) cells, MDCK cells (normal dog kidney cell line forcanine herpesvirus cultivation), CRFK cells (normal cat kidney cell linefor feline herpesvirus cultivation), CV-1 cells (African monkey kidneycell line used, for example, to culture raccoon poxvirus), COS (e.g.,COS-7) cells, and Vero cells. Particularly preferred host cells are E.coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonellatyphimurium, including attenuated strains; Spodoptera frugiperda;Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COScells; Vero cells; and non-tumorigenic mouse myoblast G8 cells (e.g.,ATCC CRL 1246). Additional appropriate mammalian cell hosts includeother kidney cell lines, other fibroblast cell lines (e.g., human,murine or chicken embryo fibroblast cell lines), myeloma cell lines,Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK cells and/or HeLacells.

Recombinant DNA technologies can be used to improve expression of atransformed polynucleotide molecule by manipulating, for example, thenumber of copies of the polynucleotide molecule within a host cell, theefficiency with which those polynucleotide molecules are transcribed,the efficiency with which the resultant transcripts are translated, andthe efficiency of post-translational modifications. Recombinanttechniques useful for increasing the expression of a polynucleotideencoding an insect esterase, or mutant thereof, include, but are notlimited to, operatively linking polynucleotide molecules to high-copynumber plasmids, integration of the polynucleotide molecule into one ormore host cell chromosomes, addition of vector stability sequences toplasmids, substitutions or modifications of transcription controlsignals (e.g., promoters, operators, enhancers), substitutions ormodifications of translational control signals (e.g., ribosome bindingsites, Shine-Dalgarno sequences), modification of polynucleotidemolecules of the present invention to correspond to the codon usage ofthe host cell, and the deletion of sequences that destabilizetranscripts.

Compositions

Compositions useful for the methods of the present invention, or whichcomprise a polypeptide of the present invention, include excipients,also referred to herein as “acceptable carriers”. An excipient can beany material that the animal, plant, plant or animal material, orenvironment (including soil and water samples) to be treated cantolerate. Examples of such excipients include water, saline, Ringer'ssolution, dextrose solution, Hank's solution, and other aqueousphysiologically balanced salt solutions. Nonaqueous vehicles, such asfixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.Other useful formulations include suspensions containing viscosityenhancing agents, such as sodium carboxymethylcellulose, sorbitol, ordextran. Excipients can also contain minor amounts of additives, such assubstances that enhance isotonicity and chemical stability. Examples ofbuffers include phosphate buffer, bicarbonate buffer and Tris buffer,while examples of preservatives include thimerosal or o-cresol, formalinand benzyl alcohol. Excipients can also be used to increase thehalf-life of a composition, for example, but are not limited to,polymeric controlled release vehicles, biodegradable implants,liposomes, bacteria, viruses, other cells, oils, esters, and glycols.

Furthermore, the insect esterase, or mutant thereof, can be provided ina composition which enhances the rate and/or degree of degradation ofhydrophobic ester pesticides or toxins, or increases the stability ofthe polypeptide. For example, the insect esterase, or mutant thereof,can be immobilized on a polyurethane matrix (Gordon et al., 1999), orencapsulated in appropriate liposomes (Petrikovics et al. 2000a and b).The insect esterase, or mutant thereof, can also be incorporated into acomposition comprising a foam such as those used routinely infire-fighting (LeJeune et al., 1998).

As would be appreciated by the skilled addressee, the insect esterase,or mutant thereof, could readily be used in a sponge or foam asdisclosed in WO 00/64539, the contents of which are incorporated hereinin their entirety.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition comprisingan insect esterase, or mutant thereof, into an animal, plant, animal orplant material, or the environment (including soil and water samples).As used herein, a controlled release formulation comprises an insectesterase, or mutant thereof, in a controlled release vehicle. Suitablecontrolled release vehicles include, but are not limited to,biocompatible polymers, other polymeric matrices, capsules,microcapsules, microparticles, bolus preparations, osmotic pumps,diffusion devices, liposomes, lipospheres, and transdermal deliverysystems. Preferred controlled release formulations are biodegradable(i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing an insect esterase, or mutant thereof, into soil orwater which is in an area sprayed with a hydrophobic ester pesticide ortoxin. The formulation is preferably released over a period of timeranging from about 1 to about 12 months. A preferred controlled releaseformulation of the present invention is capable of effecting a treatmentpreferably for at least about 1 month, more preferably for at leastabout 3 months, even more preferably for at least about 6 months, evenmore preferably for at least about 9 months, and even more preferablyfor at least about 12 months.

The concentration of the insect esterase, or mutant thereof, (or hostcell expressing the insect esterase, or mutant thereof) that will berequired to produce effective compositions for degrading a hydrophobicester pesticide or toxin will depend on the nature of the sample to bedecontaminated, the concentration of the hydrophobic ester pesticide ortoxin in the sample, and the formulation of the composition. Theeffective concentration of the insect esterase, or mutant thereof, (orhost cell expressing the insect esterase, or mutant thereof) within acomposition can readily be determined experimentally, as will beunderstood by the skilled artisan.

Surfactants

It is envisaged that the use of a surfactant in the method of thepresent invention may liberate hydrophobic ester pesticides and/ortoxins, from any, for example, sediment in the sample. Thus increasingefficiency of the method of the present invention.

Surfactants are amphipathic molecules with both hydrophilic andhydrophobic (generally hydrocarbon) moieties that partitionpreferentially at the interface between fluid phases and differentdegrees of polarity and hydrogen bonding such as oil/water or air/waterinterfaces. These properties render surfactants capable of reducingsurface and interfacial tension and forming microemulsion wherehydrocarbons can solubilize in water or where water can solubilize inhydrocarbons. Surfactants have a number of useful properties, includingdispersing traits.

Biosurfactants are a structurally diverse group of surface-activemolecules synthesized by microorganisms. These molecules reduce surfaceand interfacial tensions in both aqueous solutions and hydrocarbonmixtures. Biosurfactants have several advantages over chemicalsurfactants, such as lower toxicity, higher biodegradability, betterenvironmental compatibility, higher foaming, high selectivity andspecificity at extreme temperatures, pH and salinity, and the ability tobe synthesized from a renewable source.

Biosurfactants useful in the bioremediation methods of the presentinvention include, but are not limited to; glycolipids such asrhamnolipids (from, for example, Pseudomonas aeruginosa), trehalolipids(from, for example, Rhodococcus erythropolis), sophorolipids (from, forexample, Torulopsis bombicola), and cellobiolipids (from, for example,Ustilago zeae); lipopeptides and lipoproteins such as serrawettin (from,for example, Serratia marcescens), surfactin (from, for example,Bacillus subtilis); subtilisin (from, for example, Bacillus subtilis),gramicidins (from, for example, Bacillus brevis), and polymyxins (from,for example, Bacillus polymyxa); fatty acids, neutral lipids, andphospholipids; polymeric surfactants such as emulsan (from, for example,Acinetobacter calcoaceticus), biodispersan (from, for example,Acinetobacter calcoaceticus), mannan-lipid-protein (from, for example,Candida tropicalis), liposan (from, for example, Candida lypolytica),protein PA (from, for example, Pseudomonas. aeruginosa); and particulatebiosurfactants such as vesicles and fimbriae from, for example, A.calcoaceticus.

Transgenic Plants

The term “plant” refers to whole plants, plant organs (e.g. leaves,stems roots, etc), seeds, plant cells and the like. Plants contemplatedfor use in the practice of the present invention include bothmonocotyledons and dicotyledons. Exemplary monocotyledons include wheat,barley, rye, triticale, oats, rice, and the like.

Transgenic plants, as defined in the context of the present inventioninclude plants (as well as parts and cells of said plants) and theirprogeny which have been genetically modified using recombinant DNAtechniques to either i) cause the production of an the insect esterase,or mutant thereof, in the desired plant or plant organ.

Several techniques exist for introducing foreign genetic material into aplant cell. Such techniques include acceleration of genetic materialcoated onto microparticles directly into cells (see, for example, U.S.Pat. No. 4,945,050 and U.S. Pat. No. 5,141,131). Plants may betransformed using Agrobacterium technology (see, for example, U.S. Pat.No. 5,177,010, U.S. Pat. No. 5,104,310, U.S. Pat. No. 5,004,863, U.S.Pat. No. 5,159,135). Electroporation technology has also been used totransform plants (see, for example, WO 87/06614, U.S. Pat. Nos.5,472,869, 5,384,253, WO 92/09696 and WO 93/21335). In addition tonumerous technologies for transforming plants, the type of tissue whichis contacted with the foreign genes may vary as well. Such tissue wouldinclude but would not be limited to embryogenic tissue, callus tissuetype I and II, hypocotyl, meristem, and the like. Almost all planttissues may be transformed during development and/or differentiationusing appropriate techniques described herein.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Examples of plant promoters include, but are not limited toribulose-1,6-bisphosphate carboxylase small subunit, beta-conglycininpromoter, phaseolin promoter, ADH promoter, heat-shock promoters andtissue specific promoters. Promoters may also contain certain enhancersequence elements that may improve the transcription efficiency. Typicalenhancers include but are not limited to Adh-intron 1 and Adh-intron 6.

Constitutive promoters direct continuous gene expression in all cellstypes and at all times (e.g., actin, ubiquitin, CaMV 35S). Tissuespecific promoters are responsible for gene expression in specific cellor tissue types, such as the leaves or seeds (e.g., zein, oleosin,napin, ACP, globulin and the like) and these promoters may also be used.Promoters may also be active during a certain stage of the plants'development as well as active in plant tissues and organs. Examples ofsuch promoters include but are not limited to pollen-specific, embryospecific, corn silk specific, cotton fiber specific, root specific, seedendosperm specific promoters and the like.

Under certain circumstances it may be desirable to use an induciblepromoter. An inducible promoter is responsible for expression of genesin response to a specific signal, such as: physical stimulus (heat shockgenes); light (RUBP carboxylase); hormone (Em); metabolites; and stress.Other desirable transcription and translation elements that function inplants may be used.

In addition to plant promoters, promoters from a variety of sources canbe used efficiently in plant cells to express foreign genes. Forexample, promoters of bacterial origin, such as the octopine synthasepromoter, the nopaline synthase promoter, the mannopine synthasepromoter; promoters of viral origin, such as the cauliflower mosaicvirus (35S and 19S) and the like may be used.

The following examples are offered for illustration purposes, and arenot intended to limit or define the invention in any manner.

EXAMPLES Example 1 Construction of Mutants

An alignment of the amino acid sequence of the E3 enzyme with that of avertebrate acetylcholinesterase (TcAChE, for which the three dimensionalstructure is known; Sussman et al., 1991) is given in FIG. 1. Mutants ofE3 and EST23 were constructed using the QuickChange™ Site-DirectedMutagenesis Kit of Stratagene and are named according to the number ofthe residue that has been changed, and the nature of that change. Forexample, mutant E3W251L is an E3 mutant in which the Trp residue atposition 251 in the wild-type enzyme (i.e. E3WT) has been mutated toLeu.

E3 and EST23 enzymes were expressed using the baculovirus expressionsystem as described by Newcomb et al. (1997), but using the HyQSFX-insect serum-free medium (HyClone) for increased expression. Cellextracts were prepared by lysing the cells at a concentration of 10⁸cells ml⁻¹ in 0.1M phosphate buffer pH 7.0 containing 0.05% TritonX-100. Extracts were then titrated for the number of esterase moleculesusing a fluorometric assay based on the initial release of coumarin (afluorescent compound) upon phosphorylation of the enzyme bydiethylcoumaryl phosphate (dECP).

FIG. 2 illustrates the proposed configuration of the active site of E3(based on the three dimensional structure of vertebrate AChE) in anacylation reaction. We have examined mutations in seven E3 residues inregions corresponding to three distinct subsites of the known AChEactive site. These are the oxyanion hole (E3 residue 137), the anionicsite (E3 residues 148, 217 and 354) and acyl binding pocket (E3 residues250, 251 and 309). The anionic site and acyl binding pocket correspondto the p1 and p2 subsites in the nomenclature of Jarv (1984).

Mutations in the Oxyanion Hole

In TcAChE the oxyanion hole comprises Gly118, Gly119 and Ala201, whichcorresponds to Gly136, Gly137 and Ala219 in E3. These residues arehighly conserved throughout the carboxyl/cholinesterase multigene family(Oakeshott et al., 1999) and there is empirical evidence for theconservation of the oxyanion hole structure from X-ray crystallographicstudies of several cholinesterases and lipases (Cygler and Schrag,1997), albeit the structure does change during interfacial activation insome lipases (Derewenda et al., 1992). There is also empiricalstructural evidence for their function in stabilising the oxyanionformed by the carbonyl oxygen of the carboxylester substrate as thefirst transition state during catalysis (Grochulski et al., 1993;Martinez et al., 1994). This stabilisation is achieved by a network ofhydrogen bonds to the amide groups of the three key residues in thepeptide chain (Ordentlich et al., 1998). Recently Koellner et al. (2000)have also shown that both Gly residues in the AChE oxyanion hole makehydrogen bonds with buried “structural” water molecules, which areretained during catalysis and thought to act as lubricants to facilitatetraffic of substrates and products within the active site.

Three further mutations were made to the Gly137 of E3 in addition to theG137D found naturally in OP resistant L. cuprina. First, Glu wassubstituted as the other acidic amino acid, in G137E. The mutant G137Hwas also constructed, because His is also non-protonated at neutral pH(pK_(a) about 6.5 cf 4.4 for Asp and Glu) and it was found to confersome OP hydrolysis on human butyrylcholinesterase when substituted foreither Gly in its oxyanion hole (Broomfield et al., 1999). Finally, Arg(pK_(a) around 12) was substituted at position 137, to examine theeffects of the most strongly basic substitution possible.

Mutations in the Acyl Binding Pocket

The acyl binding pockets of structurally characterised cholinesterasesare formed principally from four non-polar residues, three of which aregenerally also aromatic. Together they create a strongly hydrophobicpocket to accommodate the acyl moiety of bound substrate. The fourresidues in TcAChE are Trp233, Phe288, Phe290 and Val400 correspondingto Trp251, Val307, Phe309 and Phe422 in E3. Similar arrays ofhydrophobic residues appear to be conserved at the corresponding sitesof most carboxyl/cholinesterases (Oakeshott et al., 1993; Robin et al.,1996; Yao et al., 1997; Harel et al., 2000). In particular Trp isstrongly conserved at residue 233/251 and 290/309 is Phe incholinesterases and most carboxylesterases, albeit a Leu or Ile inseveral lipases and a few carboxylesterases. The residue correspondingto TcAChE Phe288 is typically a branched chain aliphatic amino acid incholinesterases that show a preference for longer chain esters such asbutyrylcholine. This includes mammalian butyrylcholinesterase and someinsect acetylcholinesterases, which have a butyrylcholinesterase-likesubstrate specificity. The branched chain aliphatic amino acid appearsto provide a greater space in the acyl-binding pocket to accommodate thelarger acyl group.

Mutational studies of 288/307 and 290/309 in several cholinesterasesconfirm their key role in determining aspects of substrate specificitiesrelated to acyl group identity. In human AChE replacement of the Phe ateither position with a smaller residue like Ala improves the kinetics ofthe enzyme for substrates like propyl- or butyl-(thio)choline withlarger acyl groups than the natural acetyl (thio)choline substrate(Ordentlich et al., 1993). In AChE from D. melanogaster and thehousefly, Musca domestica, natural mutations of their 290/309 equivalentto the bulkier, polar Tyr that contributes to target site OP resistancehave lower reactivity to both acetylcholine and OPs (Fournier et al.,1992; Walsh et al., 2001). For D. melanogaster AChE, substitution ofthis Phe residue with the smaller Leu gave the predicted increase in OPsensitivity, although surprisingly replacement with other small residueslike Gly, Ser or Val did not (Villatte et al., 2000).

Trp 233/251 has received much less attention in mutational studies ofcholinesterases but our prior work on E3 shows its replacement with asmaller Leu residue again increases reactivity for carboxylestersubstrates with bulky acyl moieties as in malathion, or for OPs(Campbell et al., 1998a, b; Devonshire et al., 2002). A mutation to Glyhas also been found in a homologue from the wasp, Anisopteromaluscalandrae, that shows enhanced malathion carboxylesterase (MCE) kinetics(Zhu et al., 1999) while a Ser has been found in a homologue from M.domestica that may be associated with malathion resistance (Claudianoset al., 2002). In respect of OP hydrolase activity Devonshire et al.(2002) proposed that the particular benefit of such mutations is toaccommodate the inversion about the phosphorus that must occur for thesecond hydrolysis stage of the reaction to proceed. Notably Devonshireet al. (2002) found that the k_(cat) for OP hydrolase activity ofE3W251L is an order of magnitude higher for dMUP, with its smallerdimethyl phosphate group than for dECP, which has a diethyl phosphategroup. This suggests that there remain tight steric constraints on theinversion even in a mutant with a larger acyl pocket.

We have mutated both the W251 and F309 residues of E3 as well as theP250 immediately adjacent to W251. In addition to the previouslycharacterised natural W251L mutation we have now analysed substitutionswith four other small amino acids in W251S, W251G, W251T and W251A. Adouble mutant of W251L and P250S was also analysed, because a naturalvariant of the ortholog of E3 in M. domestica with high MCE activity hasSer and Leu at positions 250 and 251, respectively. Only one F309substitution was examined, F309L, which the AChE results suggest shouldenhance MCE and OP hydrolyse activities. F309L was analysed alone and asa double mutant with W251L.

Mutations in the Anionic Site

The anionic site of cholinesterases is sometimes called the quaternarybinding site (for the quaternary ammonium in acetylcholine), or the p1subsite in the original nomenclature of Jarv (1984). It principallyinvolves Trp 84, Glu 199 and Phe 330, with Phe 331 and Tyr 130 (TcAChEnomenclature) also involved. Except for Glu 199 it is thus a highlyhydrophobic site. Glu 199 is immediately adjacent to the catalytic Ser200. The key residues are highly conserved across cholinesterases and toa lesser extent, many carboxylesterases (Oakeshott et al., 1993;Ordentlich et al., 1995; Robin et al., 1996; Claudianos et al., 2002).Except for Trp 84 (the sequence alignment in FIG. 1 shows that E3 ismissing residues corresponding to AChE residues 74-85), E3 has identicalresidues to TcAChE at the corresponding positions (217, 354 and 148,respectively). Interestingly the equivalent of Glu 199 is Gln and theequivalent of the Phe 330 is Leu in some lipases and certaincarboxylesterases, whose substrates are known to have small leavinggroups (Thomas et al., 1999; Campbell et al., 2001; Claudianos et al.,2002).

Structural and mutational studies have provided a detailed picture ofthe role of the anionic site in cholinesterase catalysis. The keyresidues form part of a hydrogen bonded network at the bottom of theactive site, with Tyr 130 and Glu 199 also sharing contact with astructural water molecule (Ordentlich et al., 1995; Koellner et al.,2000). The anionic site undergoes a conformational change when substratebinds a peripheral binding site at the lip of the active site gorge, thenew conformation accommodating the choline (leaving) group of thesubstrate and facilitating the interaction of its carbonyl carbon withthe catalytic Ser 200 (Shafferman et al., 1992; Ordentlich et al., 1995;1996). Consequently the site functions mainly in the first, enzymeacylation, stage of the reaction and, in particular, in the formation ofthe non-covalent transition state (Nair et al., 1994). Thereforemutations of the key residues mainly affect K_(m) rather than k_(cat).The interactions with the choline leaving group are mainly mediatedthrough non-polar and π-electron interactions, principally involving Trp84 and Phe 330 (Ordentlich et al., 1995).

Studies with OP inhibitors suggest that the anionic site ofcholinesterases also accommodates their leaving group but there is someevidence that part of the site (mainly Glu 199 and Tyr 130; alsopossibly Ser 226) may also then affect the reactivity of thephosphorylated enzyme (Qian and Kovach, 1993; and see also Ordentlich etal., 1996; Thomas et al., 1999).

There has been little mutational analysis of carboxylesterase sitescorresponding to the AChE anionic site but one interesting exceptioninvolves the EST6 carboxylesterase of D. melanogaster, which has a Hisat the equivalent of Glu 199. A mutant in which this His is replaced byGlu shows reduced activity against various carboxylester substrates buthas acquired some acetylthiocholine hydrolytic activity (Myers et al.,1993). The E4 carboxylesterase of the aphid, Myzus persicae, has a Metat this position and this enzyme is unusually reactive to OPs(Devonshire and Moores, 1982). However, it is not known whether the Metcontributes to the OP hydrolase activity. Similarly, a Y148Fsubstitution is one of several recorded in the E3 ortholog in an OPresistant strain (ie also G137D) of M. domestica but it is not knownwhether this change directly contributes to OP hydrolase activity(Claudianos et al., 1999).

The Y148, E217 and F354 residues in E3 have now been mutated. E217M andY148F mutations were made to test whether the corresponding mutations inthe M. persicae and M. domestica enzymes above contribute directly totheir OP reactivity. Y148F is also tested in a G137D double mutant sincethis is the combination found in the resistant M. domestica. F354 wasmutated both to a smaller Leu residue and a larger Trp, Leu commonlybeing found at this position in lipases (see above).

Example 2 Enzyme Titrations

Four 100 μl reactions were set up for each expressed esterase inmicroplate columns 1-4

plate well blank containing 0.025% Triton X-100, 0.1M phosphate bufferpH 7.0;substrate blank containing 100 μM dECP in 0.025% Triton X-100, 0.1Mphosphate buffer pH 7.0;cell blank containing 50 μl cell extract mixed 1:1 with 0.1M phosphatebuffer pH 7.0;titration reaction containing 50 μl cell extract mixed 1:1 with 0.1Mphosphate buffer pH 7.0 containing 200 μM dECP.

All components except dECP (freshly prepared at a concentration of 200μM in buffer) were placed in the wells. Several enzymes were assayedsimultaneously in a plate, and the reactions were started by adding dECPsimultaneously to the 2nd and 4th wells down a column. The interval tothe first reading (typically 1 minute) was noted for the subsequentcalculations.

The mean value for the plate well blank (A) was subtracted from allreadings before further calculations. Preliminary experiments withvarious cell extracts showed that they gave some fluorescence at 460 nmand that their addition to solutions of the assay product,7-hydroxycoumarin, quenched fluorescence by 39(±7) %. Fluorescencevalues in the titration reactions (D) were therefore corrected for thisquenching effect after subtraction of the intrinsic fluorescence of thecell extracts (C). Finally, the substrate blank (B), taken as the meanfrom all the simultaneous assays in a plate, was subtracted to give thecorrected fluorescence caused by the esterase-released coumarin. Thesecorrections were most important for cell lines expressing esterase atvery low level (<1 μmol/μl extract).

The fully corrected data were plotted as a progress curve, and theequilibrium slope extrapolated back to zero time to determine the amountof esterase, based on its stoichiometric interaction with the inhibitor(the 100 μM concentration of dECP gave full saturation of the esterasecatalytic sites of all these enzymes in 10-20 minutes). A calibrationcurve for 7-hydroxycoumarin was prepared alongside the reactions in allplates, and used to calculate molar concentration of enzyme and productformation.

FIG. 3 shows the results of representative titration experimentsperformed on cell extracts containing baculovirus expressed esterases.

Example 3 Permethrin Hydrolysis Assays

Expressed enzymes were tested for permethrin hydrolytic activity using aradiometric partition assay for acid-labelled compounds, or a TLC basedassay for those labelled in the alcohol moiety (Devonshire and Moores,1982). Features of the assays include keeping the concentration ofpermethrin below its published solubility in aqueous solution (0.5 μM),the concentration of detergent (used to extract the enzyme from theinsect cells in which it is expressed) below the critical micelleconcentration (0.02% for Triton X100), and performing the assays quickly(ie within 10-30 minutes) to minimise the substrate sticking to thewalls of the assay tubes (glass tubes were used to minimise stickiness).At these permethrin concentrations the enzyme is not saturated by thesubstrate, so K_(m) values could not be determined. However, specificityconstants (k_(cat)/K_(m)) could be calculated accurately for each of theenzymes with permethrin activity, which allows direct comparison oftheir efficiency at low substrate concentrations. The power of theanalyses was increased by separating permethrin into its cis and transisomers.

(a) Separation of Cis and Trans Isomers of Permethrin

Commercial preparations of permethrin contain four stereoisomers: 1Scis, 1R cis, 1S trans, 1R trans (FIG. 4). Preparative thin layerchromatography (TLC) on silica was used to separate the isomers into twoenantiomer pairs: 1S/1R cis and 1S/1R trans. The enantiomers could notbe separated further. Enzyme preparations could then be assayed for thehydrolysis of each enantiomer pair.

(b) Assay Protocol Pyrethroids Radiolabelled in the Acid Moiety

This assay (Devonshire and Moores, 1982) is used for permethrin isomers.It relies on incubating the expressed esterase with radiolabelledsubstrate and then measuring the radioactive cyclopropanecarboxylateanion in the aqueous phase after extracting the unchanged substrate intoorganic solvent. Based on previous experience, the best extractionprotocol utilises a 2:1 (by volume) mixture of methanol and chloroform.When mixed in the appropriate proportion with aliquots of the assayincubation, the consequent mixture of buffer, methanol and chloroform ismonophasic, which serves the purpose of stopping the enzyme reaction andensuring the complete solubilization of the pyrethroid. Subsequentaddition of an excess of chloroform and buffer exceeds the capacity ofthe methanol to hold the phases together, so that the organic phase canbe removed and the product measured in the aqueous phase. In detail, theprotocol is as follows.

Phosphate buffer (0.1M, pH 7.0) was added to radiolabelled permethrin(50 μM in acetone) to give a 1 μM solution and the assay then started byadding an equal volume of expressed esterase appropriately diluted inthe same buffer. Preliminary work had established that the concentrationof detergent (Triton X-100 used to extract esterase from the harvestedcells) in the incubation had to be below its CMC (critical micelleconcentration of 0.02%) to avoid the very lipophilic pyrethroidpartitioning into the micelles and becoming unavailable to the enzyme.Typically, the final volume of the assay was 500-1000 μl, with substrateand acetone concentrations 0.5 μM and 1%, respectively. At intervalsranging from 30 seconds to 10 minutes at a temperature of 30°, 100 μlaliquots of the incubation were removed, added to tubes containing 300μl of the 2:1 methanol chloroform mixture and vortex-mixed. The tubeswere then held at room temperature until a batch could be furtherprocessed together, either at the end of the incubation or during anextended sampling interval. After adding 50 μl buffer and 100 μlchloroform, the mixture was vortex-mixed, centrifuged and the lowerorganic phase removed with a 500 μl Hamilton syringe and discarded. Theextraction was repeated after adding a further 100 μl chloroform, andthen 200 μl of the upper aqueous phase was removed (using a pipettorwith a fine tip) for scintillation counting. It is critical to avoidtaking any of the organic phase. Since the final volume of the aqueousphase was 260 μl (including some methanol), the total counts produced inthe initial 100 μl aliquot were corrected accordingly.

Pyrethroids Radiolabelled in the Alcohol Moiety i) Type IPyrethroids—Dibromo Analogues (NRDC157) of Permethrin:

The 3-phenoxbenzyl alcohol formed on hydrolysis of these esters does notpartition into the aqueous phase in the chloroform methanol extractionprocedure. It was therefore necessary to separate this product from thesubstrate by TLC on silica (Devonshire and Mooers, 1982). In detail, theprotocol is as follows.

Incubations were set up as for the acid-labelled substrates. Thereactions were stopped at intervals in 100 μl aliquots taken from theincubation by immediately mixing with 200 μl acetone at −79° (solidCO₂). Then 100 μl of the mixture was transferred, together with 3 μlnon-radioactive 3-phenoxbenzyl alcohol (2% in acetone), on to theloading zone of LinearQ channelled silica F254 plates (Whatman). Afterdeveloping in a 10:3 mixture of toluene (saturated with formic acid)with diethyl ether, the substrate and product were located byradioautography for 6-7 days (confirming an identical mobility of theproduct to the cold standard 3-phenoxbenzyl alcohol revealed under UVlight). These areas of the TLC plate were then impregnated with Neatan(Merck) and dried, after which they were peeled from the glass supportand transferred to vials for scintillation counting. The counts werecorrected for the 3-fold dilution of the initial 100 μl by acetonebefore spotting on the silica.

ii) Type II Pyrethroids—Deltamethrin Isomers:

Preliminary experiments, in which incubations were analysed by TLC asabove, showed primarily the formation of 3-phenoxbenzoic acid, in linewith literature reports that the initial cyanohydrin hydrolysis productis rapidly converted non-enzymically to the acid. Since the TLC assay ismore protracted than the chloroform-methanol extraction procedure, thelatter (as described above for acid-labelled pyrethroids) was adopted tomeasure the 3-phenoxbenzoate anion produced from these substrates.

For all assays the molar amount of product formed was calculated fromthe known specific activity of the radiolabelled substrate. Earlyexperiments on the expressed E3WT esterase showed that the rate ofhydrolysis was directly proportional to the concentration of 1RS cis or1RS trans permethrin in the assay up to 0.5 μM, i.e. there was noaccumulation of Michaelis complex. Assays at concentrations greater than0.5 μM, which approximates the published aqueous solubility ofpermethrin, gave erratic results so precluding the measurement of K_(m)and k_(cat). Furthermore, with the racemic substrates, the rate ofhydrolysis slowed dramatically once approximately 50% of the substratehad been hydrolysed, indicating that only one of the two enantiomers (1Ror 1S present in equal amounts in a racemic mixture) was readilyhydrolysed, in line with previously published data for an esterase fromaphids (Devonshire and Moores, 1982). Assay conditions were thereforeadjusted to measure the hydrolysis of the more-readily hydrolysedenantiomer in each pair. Sequential incubation of trans permethrin withE3WT homogenates confirmed that both showed preference for the 1S transenantiomer. In all cases, the rate of hydrolysis at 0.5 μM (or 0.25 μMfor the one enantiomer in racemic substrates), together with the molaramount of esterase determined by titration with dECP, were used tocalculate the specificity constant (k_(cat)/K_(m)) since it was notpossible to separate these kinetic parameters. The same considerationsabout substrate solubility and proportionality of response to itsconcentration were assumed for all enzymes and substrates.

(c) Calculation of Specificity Constants

FIG. 5 presents the results of an experiment in which the trans- andcis-isomers of permethrin were hydrolysed by the E3W251L enzyme.

Since the rate of hydrolysis of permethrin isomers was directlyproportional to the concentration of substrate used up to 0.5 μM (i.e.there was no significant formation of Michaelis complex), it was notpossible to measure K_(m) and k_(cat) as independent parameters. Atconcentrations well below the K_(m), the Michaelis-Menten equationsimplifies to:

$v = {{\begin{matrix}k_{cat} \\K_{m}\end{matrix}\lbrack S\rbrack}\lbrack E\rbrack}$

The specificity constant (ie k_(cat)/k_(m)) can therefore be calculatedfrom the above equation using the initial hydrolysis rate (pmol/min,calculated from the known specific activity of the radiolabelledsubstrate) and the concentrations of substrate and enzyme in the assay.The diffusion-limited maximum value for a specificity constant is10⁸-10⁹ M⁻¹sec⁻¹ (Stryer, 1981).

Example 4 Malathion Hydrolysis Assays

MCE activity was assayed as described by Campbell et al. (1998), butwithout diluting the specific activity of the ¹⁴C malathion (25 mCimmol⁻¹) for enzymes that appeared to have a low K_(m). This was anend-point assay in which malathion was extracted into an organic phasewhile radiolabelled malathion carboxylic acids, the hydrolysis productsremained, in the aqueous phase. Activity was measured over the range 50nM to 1 μM to determine the K_(m) and k_(cat), and analysed bynon-linear regression using the Enzfitter 1.05 software(Elsevier-Biosoft), with graphical output to reveal any deviation fromMichaelis-Menten kinetics. Specificity constants were calculateddirectly from the K_(m) and k_(cat) values.

Example 5 Permethrin Hydrolytic Activity of E3 and EST23 Variants

Table 2 summarises the kinetic data obtained for eighteen E3 and threeEST23 variants using cis- and trans-permethrin as substrates. Themalathion hydrolytic activity of the enzymes is also given forcomparison. In each case the data represent the hydrolysis of theenantiomer that is hydrolysed the fastest out of each of the 1S/1R cisand 1S/1R trans isomer pairs (see above).

The E3WT enzyme found in OP susceptible blowflies, and its EST23 D.melanogaster orthologue, showed significant levels of permethrinhydrolytic activity, which was specific for the trans isomers. Thewild-type enzymes showed at least an order of magnitude higher activityfor malathion (although this high MCE activity does not confer malathionresistance on the blowfly because the enzyme is readily inhibited by themalaoxon produced in vivo by the fly; Campbell et al., 1998). Mutationsin either the acyl binding pocket or anionic site regions of the activesite of the E3 enzyme resulted in significant increases in activity forboth the trans and cis isomers of permethrin. These increases inpermethrin hydrolysis were not in the main correlated with increases inmalathion hydrolytic activity.

a) Oxyanion Hole Mutations

The E3G137D mutation is responsible for diazinon resistance in the sheepblowfly. In this mutant the very small, aliphatic, neutral Gly residuein the oxyanion hole region of the active site of the enzyme is replacedby an acidic Asp, allowing hydrolysis of a bound oxon OP molecule.However, this mutant (and its D. melanogaster orthologue) had reducedactivity for trans-permethrin in particular, compared to that of thewild-type enzyme. This activity was not increased by substitution ofGly-137 with either His or Glu. However, substitution of Gly-137 withArg did not affect the activity for either cis- or trans-permethrinappreciably. The linear nature of Arg might mean that it can fold easilyand not interfere with binding of permethrin to the active site. The MCEactivity of this group of mutants correlated broadly with their activityfor trans permethrin in particular, indicating effects of G137substitutions on the accommodation and stabilisation of the substrateacyl group. Effects are generally smaller for permethrin than malathionbut this is consistent with the somewhat smaller acyl group forpermethrin.

b) Acyl Binding Pocket Mutations

The E3W251L mutation, which replaces the large aromatic Trp reside withthe smaller aliphatic Leu in the acyl pocket of the active site,resulted in a 7-fold increase in trans-permethrin hydrolysis and theacquisition of substantial cis-permethrin hydrolysis. This is themutation responsible for the acquisition of malathion resistance in thesheep blowfly. The MCE activity of this mutant was 2-fold higher thanthat of the wild-type enzyme. The effect of W251L in EST23 wasessentially the same as for E3. Replacement of Trp-251 with even smallerresidues in E3 (Thr, Ser, Ala and Gly in decreasing order of size) alsoresulted in an increase in permethrin hydrolytic activity, although theactivity of these mutants was not as high as that of E3W251L. Clearly,steric factors are not the only consideration in the activity of themutants. For example, Thr and Ser both contain hydroxyl groups and arehydrophilic. Furthermore, Ala is both aliphatic and hydrophobic (likeLeu) and even smaller than Leu, yet this mutant was as active forpermethrin as the W251L mutant. Opening up the oxyanion hole of theW251L mutant (ie E3P250S/W251L) also decreased its activity for bothcis- and trans-permethrin, although the activity was still higher thanthat of the wild type. It is interesting to note that increases inspecificity constants for permethrin for all W251 mutants in E3 as wellas W251L in EST23 compared to those of the wild types were uniformlymore pronounced for the cis isomers. Whereas the wild type enzymesyielded trans:cis ratios of at least 20:1, these ratios were only 2-6:1for the W251 mutants. The extra space in the acyl pocket provided bythese mutants was apparently of greatest benefit for the hydrolysis ofthe otherwise more problematic cis isomers.

The MCE activity of the E3-251 mutants was not correlated withpermethrin hydrolytic activity. Of this group of mutants, E3W251G hadapproximately 10-fold higher MCE activity than the remainder of thegroup, and yet its permethrin hydrolytic activity was among the lowest.

Combination of both the W251L and G137D mutations on to the same E3molecule increased the activity of the enzyme for cis permethrin overwild-type levels, but decreased the activity for trans-permethrin andalso malathion. However, the activity of the double mutant was not asgreat as that of the mutant containing the E3W251L mutation alone (i.e.the mutations did not act additively).

Some lipases are known to have a Leu residue at the positioncorresponding to Phe 309 in L. cuprina E3. The E3F309L mutant wastherefore constructed with the aim of conferring activity for lipophilicsubstrates like pyrethroids. As can be seen from Table 2, the E3F309Lmutant was much better than E3WT for both isomers. It was even moreactive for trans-permethrin than E3W251L, though not as active for thecis isomers. However, the MCE activity of this mutant was less than halfthat of the wild-type enzyme. Combination of both the F309L and W251Lmutations on the same E3 molecule increased the activity forcis-permethrin and decreased the activity for trans-permethrin toE3W251L levels. In other words, the F309L mutation had very littleeffect on the activity of the W251L mutant for permethrin, but decreasedits activity for malathion.

c) Anionic Site Mutations

Some lipases are known to have a Leu residue at the positioncorresponding to Phe 354 in L. cuprina E3. However, substitution of Phe354 for Leu in E3 did not increase its activity for permethrinappreciably, but greatly reduced its activity for malathion.Substitution of Phe 354 for the bulkier aromatic residue, Trp, on theother hand, increased activity for both cis- and trans-permethrin3-4-fold, but decreased MCE activity slightly. It is perhaps surprisingthat F354W, not F354L, should show increases in activity against thevery lipophilic permethrin, given that it is a Leu that replaces Phe insome naturally occurring lipases.

Although Y148F is of little consequence for MCE activity it has largeeffects on permethrin kinetics and the effects are opposite in directiondepending on genetic background. As a single mutant compared to wildtype it shows 5-6 fold enhancement of activity for both cis and transpermethrin. As a double mutant with G137D (which as a single mutantgives values much lower than wild type), it shows a further two foldreduction for trans permethrin and almost obliterates activity for cispermethrin. These latter results clearly imply a strong interaction ofY148 with the oxyanion hole in respect of permethrin hydrolysis.

Glu-217, the residue immediately adjacent to the catalytic serine, isthought to be important in stabilising the transition state intermediatein hydrolysis reactions. However, mutating this residue to Met(E3E217M), as found naturally in the esterase E4 of the aphid M.persicae, had little effect on permethrin activity but greatly reducedits MCE activity.

Example 6 Hydrolysis of Bromo-Permethrin Analogue

Table 2 also summarises the kinetic data obtained for the E3 and EST23variants using the two cis-dibromovinyl analogues of permethrin(NRDC157). The 1S cis isomer of this dibromo analogue of permethrin washydrolysed with similar efficiency to the 1R/1S cis permethrin by allenzymes except E3F309L and F309L/W251L. This indicates that the largerbromine atoms did not substantially obstruct access of this substrate tothe catalytic centre. Although the activities with the E3WT and EST23WTenzymes were too low for significant comparison between isomers, allother enzymes except E3F309L and F309L/W251L showed 10 to 100-foldfaster hydrolysis of the 1S isomer. This is the same preference for thisconfiguration at C1 of the cyclopropane ring as found previously for 1Strans permethrin in M. persicae (Devonshire and Moores, 1982).

F309L showed a dramatic effect on NRDC157 kinetics. The single mutantshowed little difference from wild type for 1S cis and the double withW251L showed less activity than W251L alone for this isomer. However,the 1S/1R preference was reversed, with values of 0.7:1 in the singlemutant and 0.4:1 in the double. The result is the two highest values for1R cis activities in all the data set. The value for the double mutantis in fact about 10 fold higher than those for either mutant alone.

Example 7 Hydrolysis of Type II Pyrethroids by Expressed Enzymes

Table 3 summarises the kinetic data obtained for a sub-set of the E3 andEST23 variants using the four deltamethrin cis isomers. With theexception of E3W251L and E3F309L, the 1R cis isomers of deltamethrin(whether αS or αR) were hydrolysed with similar efficiency to the 1R cisNRDC157 (which can be considered intermediate in character betweenpermethrin and deltamethrin in that it has dibromovinyl substituent butlacks the α cyano group). Activity against 1R cis isomers was alwaysgreater with the αR than the αS conformation. E3W251L and E3F309L weremarkedly less efficient with the 1R cis isomers of deltamethrin thanwith the corresponding isomers of NRDC157.

TABLE 2 Specificity constants of natural and synthetic variants of L.cuprina esterase E3 and D. melanogaster EST23 for the cis- andtrans-isomers of permethrin, malathion and the two cis-dibromovinylanalogues of permethrin (NRDC157). Specificity Constant (k_(cat)/K_(m)M⁻¹ sec⁻¹) 1S/1R 1S/1R cis- NRDC157 trans- permethrin NRDC157 1R cisEnzyme permethrin (trans:cis ratio) malathion 1S cis (1S:1R ratio) E3WT 90 000 3 400 (27:1) 2 600 000  4 700 630 (8:1) Oxyanion hole mutants:E3G137D  9 600 1 800 (5:1)   5 100  ND¹ ND E3G137R  85 000 3 900 (22:1)1 200 000 ND ND E3G137H  26 000 1 600 (16:1)   8 800 ND ND E3G137E  2400 280 (9:1)   19 000 ND ND Acyl binding pocket mutants: E3W251L 900000 460 000 (2:1) 4 800 000 370 000  5 400 (68:1) E3W251S 140 000 36 000(4:1) 6 500 000 35 000 2 900 (12:1) E3W251G  95 000 24 000 (4:1) 57 000000  27 000 1 700 (16:1) E3W251T 150 000 24 000 (6:1) 4 500 000 24 000900 (26:1) E3W251A 300 000 72 000 (4:1) 5 400 000 67 000 1 200 (56:1)E3F309L 1 200 000  48 000 (25:1) 1 000 000  5 700 8 000 (0.7:1) E3W251L/810 000 430 000 (2:1) 1 400 000 26 000 69 100 (0.4:1) F309L E3W251L/  24000 11 000 (2:1)   60 000 12 000 1 100 (11:1) G137D E3P250S/ 340 000 110000 (3:1) 1 400 000 ND ND W251L Anionic site mutants: E3Y148F 580 000 17000 (34:1) 3 100 000 ND ND E3Y148F/  4 100 47 (87:1)   12 000 ND NDG137D E3E217M  93 000 4 400 (21:1)   77 000 ND ND E3F354W 350 000 8 800(40:1) 1 600 000 ND ND E3F354L 104 400 2 700 (38:1)  106 000 ND ND . . .EST23 enzymes: EST23WT  21 000 890 (24:1) 2 700 000   990 330 (3:1)EST23W251L 260 000 160 000 (2:1) 2 300 000 72 000 1 200 (60:1)EST23G137D  2 500 —² — ND ND Ratios of the specificity constants fortrans and cis permethrin, and for 1S cis and 1R cis NRDC157 are alsoindicated. ¹Not determined ²Not substantially different from valuesobtained using control cell extracts

Significantly, the 251 mutant with the highest deltamethrin activitieswas W251S, while W251L (highest for the other two pyrethroids), andW251G (highest for malathion) gave the lowest deltamethrin activities ofthe five 251 mutants. This suggests that accommodation of the αcyanomoiety of the leaving group may be the major impediment to efficientdeltamethrin hydrolysis, sufficient to prevent any significanthydrolysis by wild type E3. Accommodation of substrate requiressignificantly different utilisation of space across the active sitecompared to other substrates, such that substitution of W251 in the acylpocket with a smaller residue allows useful accommodation, particularlyfor αR isomers. Importantly, however, the details of the spatialrequirements, and therefore the most efficacious mutants, differ fromthose for the other pyrethroids.

The activity of all enzymes with the 1S cis isomers of deltamethrin wasdramatically less than with the corresponding isomer of NRCC157 lackingthe α cyano group. This dramatic influence of the μ cyano group appearsto be expressed with this 1S conformation at C1 of the cyclopropanegroup. With the exception of some of the least active mutants, activityagainst 1S cis isomers was again always greater with the αR than the αSconformation.

Example 8 General Discussion

Together, the permethrin and NRDC157 results for the 251 series mutantsgenerate some quite strong and simple inferences about acyl bindingconstraints in E3/EST23. Overall, as with malathion, 251 replacementsthat should generate a more spacious acyl pocket do facilitate theaccommodation/stabilisation of the bulky acyl groups of thesesubstrates. These replacements are beneficial to the hydrolysis of allthe isomers generated by the two stereocentres across the cyclopropanering. While the trans isomers are strongly preferred by wild typeenzyme, the mutants can also hydrolyse at least part of the cis isomermix relatively well. However, within the cis isomers the improvements inthe mutants is much more marked for the 1S cis isomers. The 1R cisisomers, which are the most problematic of all configurations for wildtype enzyme, remain the most problematic for the mutants. Within themutant series, the improved kinetics are not simply explained by thereduction in side chain size; the smallest substitution does not givethe highest activities, as it does for malathion. Indeed the bestkinetics are obtained with W251L, although Leu has the greatest sidechain size of all the replacements tested.

TABLE 3 Specificity constants for the four deltamethrin cis isomersSpecificity Constant (k_(cat)/K_(m) M⁻¹sec⁻¹) 1S cis αR 1S cis αS 1R cisαR 1R cis αS Enzyme deltamethrin deltamethrin deltamethrin deltamethrinE3WT  —¹ — — — E3G137D — —  890 560 E3G137R — —  670 350 E3G137H ND NDND ND E3G137E ND ND ND ND E3W251L  990 880  380 — E3W251S 4 600 2 460  ND² ND E3W251G  700 170  690 350 E3W251T 2 900 520 2 100 1 300  E3W251A2 000 660 1 300 730 E3F309L 2 400 810 1 600 840 E3W251L/ 3 600 410 2 7001 100  G137D Est23WT  450 750 — — Est23W251L  980 550  1000 430 ¹Notsubstantially different from values obtained using control cell extracts²Not determined

In contrast to the relatively simple and consistent patterns seen forpermethrin and NRDC157, the deltamethrin results for the 251 seriesmutants are quite complex and difficult to interpret. As might beexpected from their enhanced kinetics for the other substrates, they doshow overall better activities than wild type for the four cisdeltamethrin isomers, albeit as with wild type they are much lower inabsolute terms than for the other substrates. However, the preferencefor 1S over 1R isomers, which is so strong in respect of NRDC157, isweak at best in the deltamethrin data. On the other hand there is aclear trend across all the mutants for a preference for the αR over αSisomers. It is generally only of the order of 2:1, but notably it isopposite to the trend shown by wild type EST23. It is at first sightunexpected that these presumptive acyl binding pocket replacementsshould affect αR/αS stereopreferences because the latter apply to theα-cyano moiety in the (alcohol) leaving group of the substrate.

Overall the F309L data clearly show a major effect of this residue onthe kinetics of pyrethroid hydrolysis. At one level there are parallelswith the results for the W251 series mutants, both data sets showingenhanced kinetics consistent with expectations based on the provision ofgreater space in the acyl binding pocket. However, there are alsoimportant differences, with the W251 series disproportionately activefor the cis vs. trans isomers of permethrin and F309L disproportionatelyactive with 1R vs. 1S isomers of cis NRDC157. The replacements at thetwo sites also show strong interactions, consistent with themcontributing to a shared structure and function in the acyl bindingpocket. For example, both the disproportionate enhancement of the W251mutants for cis permethrin and the disproportionate enhancement of F309Lfor 1R cis NRDC157 behave as dominant characters in the double mutant.The 251 and 309 mutants have quantitatively similar enhancing effects onactivities and the same stereospecificities in respect of deltamethrinhydrolysis and the stereospecific differences seen with the smallerpyrethroids are not seen. However, we argue that the additional bulk ofthe αcyano moiety in its leaving group requires such a radicalreallocation of space across the active site that thestereospecificities evident with the smaller pyrethroids are overridden.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed above are incorporated herein in theirentirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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1. A method of eliminating or reducing the concentration of ahydrophobic ester pesticide or toxin in a sample, the method comprisingcontacting the sample with an insect esterase, or a mutant thereof. 2.The method of claim 1, wherein the insect esterase is anα-carboxylesterase.
 3. The method of claim 1, wherein the mutant insectesterase is an α-carboxylesterase, and has a mutation(s) in an oxyanionhole, acyl binding pocket or anionic site regions of an active site ofthe esterase, or any combination thereof.
 4. The method of claim 3,wherein the mutant insect esterase is selected from the group consistingof E3G137R, E3G137H, E3W251L, E3W251S, E3W251G, E3W251T, E3W251A,E3W251L/F309L, E3W251L/G137D, E3W251L/P250S, E3F309L, E3Y148F, E3E217M,E3F354W, E3F354L, and EST23W251L.
 5. The method of claim 2, wherein theα-carboxylesterase, or mutant thereof, comprises a sequence selectedfrom the group consisting of i) a sequence as shown in SEQ ID NO:1, ii)a sequence as shown in SEQ ID NO:2, and iii) a sequence which is atleast 40% identical to i) or ii) which is capable of hydrolysing ahydrophobic ester pesticide or toxin.
 6. The method of claim 6, whereinthe sequence is at least 80% identical to i) or ii).
 7. The method ofclaim 6, wherein the sequence is at least 90% identical to i) or ii). 8.The method of claim 1, wherein the method is performed using two or moreinsect esterases, or mutants thereof.
 9. The method of claim 1, whereinthe hydrophobic ester pesticide or toxin is a pyrethroid.
 10. The methodof claim 9, wherein the pyrethroid is a Type I or Type II pyrethroid.11. The method of claim 10, wherein the Type I pyrethroid is selectedfrom the group consisting of 1S/1R trans permethrin, 1S/1R cispermethrin, NRDC157 1S cis, and NRDC157 1R cis.
 12. The method of claim10, wherein the Type II pyrethroid is deltamethrin.
 13. The method ofclaim 1, wherein the method is performed in a liquid containingenvironment.
 14. The method of claim 1, wherein the insect esterase, ormutant thereof, is provided directly to the sample.
 15. The method ofclaim 1, wherein the insect esterase, or mutant thereof, is provided tothe sample by expression of a polynucleotide encoding the insectesterase, or mutant thereof, from a host cell comprising thepolynucleotide.
 16. The method of claim 1, wherein the insect esterase,or mutant thereof, is provided as a polymeric sponge or foam, the foamor sponge comprising the insect esterase, or mutant thereof, immobilizedon a polymeric porous support.
 17. The method of claim 1, wherein themethod further comprises the presence of a surfactant when thehydrophobic ester pesticide or toxin is contacted with the insectesterase, or mutant thereof.
 18. The method of claim 17, wherein thesurfactant is a biosurfactant.